Detection of enzyme activity is one of the major goals of molecular imaging to aid in the assessment of tumor aggressiveness tumor phenotyping to select the appropriate molecular therapeutic, and to monitor therapeutic response. Ultrasound, the most versatile and most commonly used imaging modality that boasts high spatial and temporal resolution as well as high sensitivity to microbubbles, has been limited to the detection of intravascular targets. This project aims to bring to ultrasound the ability to detect enzymes. We have successfully produced emulsions and nanobubbles with particles less than 100nm that are capable of exiting the vascular space. We propose to use two populations of particles where each carries one of the pairs of an adhesion molecule pair, such as biotin-avidin, whose ability to interact is blocked until exposed to the enzyme of interest. The enzyme activates the interaction to produce an aggregate of particles that becomes trapped in the tumor. Because ultrasound backscatter is related to the scatterer's radius raised to the 6th power, signal increases dramatically allowing aggregate detection. To avoid the use of biotion-avidin because of the known allergic effects, we selected complimentary DNA strands as the adhesion pair because they rapidly form a tight double helix when exposed to each other. To prevent plasma degradation and to block interaction, each DNA strand is cyclized with a peptide that can be cleaved by the enzyme of interest. To accomplish this we propose 5 aims that proceed with increasing complexity. We will first use liquid fluorocarbon emulsions and then proceed to the more fragile nanobubbles: 1) We will first prove in-vitro using DNA strands cyclized by a disulfide bond that are opened by TCEP, a reducing agent, that when the strands open aggregation occurs and ultrasound signal increases. 2) We will then cyclize the DNA with a peptide linker cleavable by thrombin and as in Aim I, prove that signal increases in vitro in the presence of thrombin and then in vivo using an acute thrombus that provides an intravascular target. 3) We will repeat Aim I, but the peptide linker will be replaced by a peptide cleavable by matrix metalloproteinase (MMP). We will then prove signal increase in vivo using mice with an MMP+ or MMP- tumor. 4) We will then repeat Aims 1-3 using nanobubbles after they have been optimized to maximize signal difference between their non-aggregated and aggregated state. 5) For aims 1 to 4 the DNA strands were attached to the particles using biotin/avidin linkers for convenience. In parallel with Aims 1-4 we will work to attach the DNA strands directly to the shell to increase the probability of translating the agent to the clinic. [unreadable] [unreadable] [unreadable]